Beta-amylase, gene coding therefor and manufacturing method thereof

ABSTRACT

To provide novel β-amylase derived from a microorganism and a gene thereof. β-amylase derived from  Bacillus flexus  is provided.

TECHNICAL FIELD

The present invention relates to a novel β-amylase. More particularly,the present invention relates to a β-amylase derived from amicroorganism, a gene thereof, and a manufacturing method thereof.

BACKGROUND ART

Conventionally, β-amylase of plant origin, for example, β-amylase fromsoybeans, wheat, barley, malt, a sweet potato, and a potato has beenknown. Among them, β-amylase extracted and purified from grains such assoybeans, wheat, barley, and malt is industrially widely used formanufacturing, for example, maltose-containing syrup used in sugarproduction, bakery, and brewing industries. Among the β-amylase of plantorigin, β-amylase derived from soybeans has a high enzymatic activityand also an excellent thermo stability.

By the way, in recent years, due to the increase in demand forbioethanol, the price of corn has risen. Consequently, planting has beenshifted from soybeans or wheat to corn. Therefore, soybeans, wheat,barley, and the like, are in short supply, and the prices thereof arerising. Under such circumstances, it is difficult to secure rawmaterials of β-amylase.

β-amylase is an enzyme that acts on polysaccharides such as starch andglycogen, which have the α-1,4 linkage of glucose as a main chain, andbreaks down them in a maltose unit from the non-reducing end. β-amylasehas traditionally been known to be found in higher plants such assoybeans and wheat. Since 1972 when it was reported that an enzymeexhibiting the action mechanism the same as that of higher plantβ-amylase was present also in microorganisms, a large number ofmicroorganisms have been found as β-amylase-producing microorganisms(Non-patent Document 1).

To date, Bacillus sp. such as Bacillus cereus, Bacillus polymyxa,Bacillus circulans, Bacillus megaterium, and Bacillusstearothermophilus, Streptomyces sp., Pseudomonas sp., and the like,have been reported as the β-amylase-producing microorganisms. However,most of them have low productivity, and few of them have beenpractically used.

On the other hand, amylase produced by filamentous fungi such asAspergillus sp., breaks down amylose and amylopectin by the end type.Therefore, when the amylase of this type is used, glucose, maltotriose,and other oligosaccharides, in addition to maltose, are produced.Furthermore, the amylase of this type has a low thermostability and isless practical for production of maltose.

Bacillus stearothermophilus produces a maltose-producing enzyme having ahigh thermostability (see, Patent Document 1 and Non-Patent Document 2).This enzyme produces maltose by the exo type from the non-reducing endof starch, but maltose produced is α type. Furthermore, this enzyme doesnot hydrolyze strictly in a maltose unit as in β-amylase of plantorigin. That is to say, it is reported that, in the initial time ofreaction, in addition to maltotetraose (G4), maltotriose (G3) andmaltose (G2), a small amount of maltopentaose (G5) and maltohexaose (G6)are also produced, and that this enzyme breaks down Shardinger dextrininto maltose and glucose, and breaks down maltotriose into maltose andglucose. As a result, in the starch decomposed product by this enzyme, 6to 8% glucose is contained. Therefore, this enzyme is not suitable formanufacturing highly purified maltose syrups.

Citation List [Patent Documents]

[Patent Document 1] Japanese Patent Application Unexamined PublicationNo. S60-2185

[Non-Patent Documents]

[Non-Patent Document 1] “Handbook of Industrial Sugar Enzyme,” KodanshaScientific, 1999

[Non-Patent Document 2] H. Outtrup and B. E. Norman, Starch, Vol. 12,pages 405 to 411

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

As mentioned above, it is difficult to secure stable supply of β-amylaseof plant origin that is a mainstream at the present time. Furthermore,the amount of enzyme obtained from plants is preliminarily determined,and the production amount is limited. On the other hand, few ofβ-amylase derived from microorganisms have been practically used becausethe productivity is low or mass production is difficult.

Means to Solve the problem

In view of the above-mentioned problems, the present inventors havekeenly investigated, and, as a result, have found that Bacillus flexusof Bacillus subtilis produces β-amylase having a thermostabilitycomparable to that of β-amylase derived from soybeans. Furthermore, thepresent inventors have succeeded in isolating and purifying theβ-amylase, and determining enzymological properties. Furthermore, thepresent inventors have succeeded in determining a base sequence of agene encoding the β-amylase. In addition, they have confirmed that it ispossible to manufacture β-amylase by using a transformant into which avector containing the gene has been introduced.

The present invention has been completed based on the above-mentionedresults and includes the followings.

[1] A β-amylase derived from Bacillus flexus.

[2] A β-amylase having the following enzymological properties:

(1) action: acting on the α-1,4 glucoside linkage of polysaccharides andoligosaccharides to liberate maltose;

(2) substrate specificity: acting well on starch, amylose, amylopectin,glycogen, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose,but not acting on pullulan, dextran, cyclodextrin, and maltotriose;

(3) optimum temperature: about 55° C.;

(4) optimum pH: about 8.0;

(5) thermostability: stable at 55° C. or lower (pH 5.0, 10 minutes);

(6) pH stability: stable at pH 4 to 9 (30° C., three hours); and

(7) molecular weight: about 60,000 (SDS-PAGE).

[3] A β-amylase having an amino acid sequence set forth in SEQ ID NO: 7,or an amino acid sequence equivalent to the amino acid sequence.

[4] The β-amylase described in [3], wherein the equivalent amino acidsequence is an amino acid sequence having about 90% or more identity tothe amino acid sequence set forth in SEQ ID NO: 7.

[5] An enzyme preparation including β-amylase described in any one of[1] to [4] as an active ingredient.

[6] A β-amylase gene including DNA selected from the group consisting ofthe following (A) to (C):

(A) DNA encoding an amino acid sequence set forth in SEQ ID NO: 7;

(B) DNA having a base sequence set forth in SEQ ID NO: 6; and

(C) DNA having a base sequence equivalent to the base sequence set forthin SEQ ID NO: 6, and having a β-amylase activity.

[7] A recombinant vector containing a β-amylase gene described in [6].

[8] A transformant into which a β-amylase gene described in [6] isintroduced.

[9] A manufacturing method of β-amylase, the method including thefollowing steps (1) and (2) or steps (i) and (ii):

(1) culturing Bacillus flexus having an ability of producing β-amylase:

(2) collecting β-amylase from a culture solution and/or a cell bodyafter culturing;

(i) culturing a transformant described in [8] under the conditions inwhich a protein encoded by the gene is produced; and

(ii) collecting the produced protein.

[10] The manufacturing method described in [9], wherein Bacillus flexusis a strain specified by the accession number NITE BP-548.

[11] A Bacillus flexus strain specified by the accession number NITEBP-548.

[12] A production method of maltose, the method including allowingβ-amylase derived from Bacillus flexus to act on polysaccharide oroligosaccharide having α-1,4 linkage of glucose as a main chain.

[13] The production method described in [12], wherein the β-amylase is aβ-amylase described in any one of [2] to [4].

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing an optimum temperature of β-amylase derivedfrom Bacillus flexus.

FIG. 2 is a graph showing an optimum pH of β-amylase derived fromBacillus flexus. •: citric acid buffer at pH2, 3, and 4, •:Britton-Robinson buffer at pH4, 5, 6, 7, 8, 9, 10, and 11

FIG. 3 is a graph showing thermostability of β-amylase derived fromBacillus flexus.

FIG. 4 is a graph showing pH stability of β-amylase derived fromBacillus flexus. •: citric acid buffer at pH2, 3, and 4, □:Britton-Robinson buffer at pH4, 5, 6, 7, 8, 9, 10, and 11

FIG. 5 shows the results of SDS-PAGE of purified β-amylase and samplesduring purification. Lane 1: ammonium sulfate fractionation, lane 2:HiPrepButyl 16/10 FF, lane 3: HiTrap CM FF, and lane 4: HiLoad 16/60Superdex200

FIG. 6 shows a structure of an expression plasmid pET-BAF.

DESCRIPTION OF EMBODIMENT (Terms)

The term “DNA encoding protein” in the present invention denotes DNAfrom which the protein is obtained when it is expressed, that is, DNAhaving a base sequence corresponding to an amino acid sequence of theprotein. Therefore, the codon degeneracy is also taken intoconsideration.

In the present specification, the term “isolated” and “purified” areused interchangeably. The term “isolated” used with respect to theenzyme of the present invention (β-amylase), which is derived from anatural material, denotes a state in which components other than theenzyme are not substantially contained (in particular, contaminatedprotein is not substantially contained) in the natural material.Specifically, in the isolated enzyme of the present invention, thecontent of the contaminant protein is, for example, less than about 20%,preferably less than about 10%, further preferably less than about 5%,and yet further preferably less than about 1% with respect to the totalamount on the weight basis. On the other hand, the term “isolated” whenthe enzyme of the present invention is prepared by geneticallyengineering technique denotes a state in which other components derivedfrom a host cell to be used, a culture solution, and the like, are notsubstantially contained. Specifically, for example, in the isolatedenzyme of the present invention, the content of the contaminantcomponents is less than about 20%, preferably less than about 10%,further preferably less than about 5%, and yet further preferably lessthan about 1% with respect to the total amount on the weight basis.Unless otherwise specified, when merely the term “β-amylase” is used inthis specification, it signifies the “β-amylase in an isolated state.”The same is true to the term “the present enzyme” used instead ofβ-amylase.

The term “isolated” used with respect to DNA denotes typically that DNAis separated from other nucleic acid coexisting in nature when the DNAoriginally exists in nature. However, some of the other nucleic acidcomponents such as a neighboring nucleic acid sequence in nature (forexample, a sequence of a promoter region, a terminator sequence, or thelike) may be included. For example, in the “isolated” state of thegenome DNA, the isolated DNA preferably does not substantially includeother DNA components coexisting in nature. On the other hand, in the“isolated” state of DNA prepared by a genetic engineering technique, forexample, a cDNA molecule, and the like, preferably, the DNA does notsubstantially include cell components, a culture solution, or the like.Similarly, in the “isolated” state in the case of DNA prepared bychemical synthesis, the DNA does not include a precursor (a rawmaterial) or chemical materials used in synthesis, for example, dNTP.Unless otherwise specified, when merely the term “DNA” is used in thisspecification, it signifies the “DNA in an isolated state.”

(β-Amylase and Producing Microorganism Thereof)

A first aspect of the present invention provides β-amylase (hereinafter,also referred to as “the present enzyme”) and the producingmicroorganism thereof. As shown in the below-mentioned Example, thepresent inventors have keenly investigated, and, as a result, have foundthat Bacillus flexus produces thermostable β-amylase. Furthermore, thepresent inventors have succeeded in isolation and purification, and alsosucceeded in determination of its enzymological property.

(1) Action

The present enzyme is β-amylase, and it acts on the α-1,4 glucosidelinkage in polysaccharides and oligosaccharides to liberate maltose. Thepresent enzyme hardly liberates glucose.

(2) Substrate Specificity

The present enzyme is excellent in the substrate specificity and wellacts on starch, amylose, amylopectin, glycogen, maltotetraose,maltopentaose, maltohexaose, and maltheptaose. On the contrary, thepresent enzyme does not act on pullulan, dextran, cyclodextrin, andmaltotriose.

When the present enzyme has a relative activity of 50% or more withrespect to the basic activity (100%) that is a value when a solublestarch is used as a substrate, the substrate is determined to be a“substrate on which an enzyme acts on well.” Similarly, when therelative activity is less than 10%, the substrate is determined to be a“substrate on which the enzyme does not act.” The enzyme does notsubstantially act on maltotriose and cyclodextrin (α, β, or γ).

Note here that the reactivity and the substrate specificity of thepresent enzyme can be measured and evaluated by the method shown in thebelow-mentioned Examples (see, column of a measurement method of theβ-amylase activity).

(3) Optimum Temperature

The optimum temperature of the present enzyme is about 55° C. Thepresent enzyme shows high activity at a temperature in the range fromabout 50° C. to about 60° C. The optimum temperature is a valuecalculated by the below-mentioned measurement method of β-amylaseactivity (0.1 M phosphate-HCl buffer solution (pH 5.0)).

(4) Optimum pH

The optimum pH of the present enzyme is about 8.0. The present enzymeshows high activity in the range from pH about 6.0 to about 9.0. Theoptimum pH is determined, for example, based on the results of themeasurement in a citric acid buffer with respect to pH region of pH 2 to4, and based on the results of the measurement in a Britton-Robinsonbuffer with respect to pH region of pH 4 to 11.

(5) Thermostability

The present enzyme shows an excellent thermostability that is comparableto that of β-amylase derived from soybeans. The present enzyme maintains90% or more of the activity in 0.1M acetic acid-hydrochloric acid buffersolution (pH 5.0) containing 10 mM calcium acetate at 55° C. for 10minutes.

(6) pH Stability

The enzyme shows stable activity in such a wide pH range as pH 4 to 9.That is to say, when pH of an enzyme solution subjected to treatment iswithin the range, the enzyme shows 70% or more activity with respect tothe maximum activity after treatment at 30° C. for three hours. Theoptimum pH is determined, for example, based on the results of themeasurement in a citric acid buffer for the pH region of pH 2 to 4, andbased on the results of the measurement in a Britton-Robinson buffer forthe pH region of pH 4 to 11.

(7) Molecular Weight

The molecular weight of the enzyme is about 60,000 (by SDS-PAGE).

Preferably, the present enzyme is β-amylase derived from Bacillusflexus. Herein, the “β-amylase derived from Bacillus flexus” denotesβ-amylase produced by microorganisms classified in Bacillus flexus(which may be wild-type strain and mutant strain), or β-amylase producedby using a β-amylase gene of Bacillus flexus (which may be wild-typestrain and mutant strain) obtained by a genetic engineering technique.Therefore, the “β-amylase derived from Bacillus flexus” includes arecombinant produced by using a host microorganism into which aβ-amylase gene (or a gene obtained by modifying the gene) obtained fromBacillus flexus has been introduced.

Bacillus flexus from which the present enzyme is derived is representedby a producing microorganism of the present enzyme for easy description.Examples of the producing microorganism of the present enzyme mayinclude Bacillus flexus DSM1316 (DSMZ, Germany), DSM1320 (DSMZ,Germany), DSM1667 (DSMZ, Germany), and APC9451. The APC9451 strain isdeposited with the predetermined depositary as mentioned below, andeasily available.

Depositary institution: Department of Biotechnology, National Instituteof Technology and Evaluation (NITE), Patent Microorganisms DepositaryCenter (2-5-8, Kazusa Kamatari, Kisarazu-shi, Chiba, 292-0818, Japan)

Deposited date (accepted date): Apr. 9, 2008

Accession number: NITE BP-548

As mentioned above, the details of the property of the present enzymethat has been successfully obtained has been clarified. As a result, ithas been revealed that the present enzyme is excellent inthermostability and excellent in substrate specificity. Therefore, thepresent enzyme is useful for food processing and saccharification.

The present inventors have further investigated and, as a result, havedetermined an amino acid sequence (SEQ ID NO: 7) of β-amylase producedby Bacillus flexus. Thus, one embodiment of the present invention ischaracterized in that the present enzyme consists of a protein having anamino acid sequence set forth in SEQ ID NO: 7. Herein, in general, whena part of the amino acid sequence of a certain protein is modified, themodified protein may sometimes have a function the same as that of theprotein before modification. That is to say, the modification of theamino acid sequence does not have a substantial effect on the functionof the protein, so that the function of the protein may be maintainedbefore and after the modification. As another embodiment, the presentinvention provides a protein having an amino acid sequence equivalent tothe amino acid sequence set forth in SEQ ID NO: 7 and having theβ-amylase activity (hereinafter, which is referred to as “equivalentprotein”). The “equivalent amino acid sequence” herein denotes an aminoacid sequence that is partly different from the amino acid sequence setforth in SEQ ID NO: 7 but this difference does not have a substantialeffect on the function (herein, the β-amylase activity) of the protein.The term “having a β-amylase activity” denotes having an activity ofacting on polysaccharides or oligosaccharides of glucose such as starchand glycogen, which has the α-1,4 linkage as a main chain, thus breakingdown a maltose unit from the non-reducing end. However, the degree ofthe activity is not particularly limited as long as the function ofβ-amylase can be exhibited. However, it is preferable that the activityis equal to or higher than that of the protein having the amino acidsequence set forth in SEQ ID NO: 7.

The “partial difference in the amino acid sequence” typically denotesthat mutation (change) occurs in an amino acid sequence due to deletionor substitution of one to several amino acids constituting the aminoacid sequence, or addition or insertion of one to several amino acids,or the combination thereof. Herein, the difference in the amino acidsequence is permitted as long as the β-amylase activity is maintained(more or less change in the activity is permitted). As long as thiscondition is satisfied, the position in which a difference in the aminoacid sequence occurs is not particularly limited and the difference mayoccur in a plurality of positions. The plurality herein signifies anumerical value corresponding to less than about 30%, preferably lessthan about 20%, further preferably less than about 10%, still furtherpreferably less than about 5%, and most preferably less than about 1%with respect to the total amino acid. That is to say, the equivalentprotein has, for example, about 70% or more, preferably about 80% ormore, further preferably about 90% or more, still further preferablyabout 95% or more and most preferably about 99% or more identity to theamino acid sequence set forth in SEQ ID NO: 7.

Preferably, an equivalent protein is obtained by allowing conservativeamino acid substitution to be generated in an amino acid residue that isnot essential to the β-amylase activity. Herein, “conservative aminoacid substitution” denotes substitution of an amino acid residue to anamino acid residue having a side chain of the same property. The aminoacid residue is classified into some families according to its sidechain, for example, the basic side chain (for example, lysin, arginine,and histidine), the acid side chain (for example, asparatic acid, andglutamic acid), the uncharged polar side chain (for example, glycine,asparagine, glutamine, serine, threonine, tyrosine, and cysteine), thenonpolar side chain (for example, alanine, valine, leucine, isoleucine,proline, phenyl alanine, methionine, and tryptophane), β branched sidechain (for example, threonine, valine, and isoleucine), and the aromaticside chain (for example, tyrosine, phenyl alanine, tryptophane, andhistidine). The conservative amino acid substitution is carried outbetween the amino acid residues in the same family.

The “equivalent protein” may have an additional property. Examples ofsuch a property include a property that stability is more excellent thanthe protein including the amino acid sequence set forth in SEQ ID NO: 7,a property that function that is different only at low temperatureand/or high temperature is exhibited, and a property that an optimum pHis different.

The identity (%) between two amino acid sequences or two nucleic acids(hereinafter, referred to as “two sequences” as a term including theboth) can be determined by the following procedure. Firstly, twosequences are aligned for optimum comparison of the two sequences (forexample, a gap may be introduced into the first sequence so as tooptimize the alignment with respect to the second sequence). When amolecule (amino acid residue or nucleotide) at a specific position inthe first sequence and a molecule in the corresponding position in thesecond sequence are the same as each other, the molecules in thepositions are defined as being identical. The identity between twosequences is a function of the number of identical positions shared bythe two sequences (i.e., identity (%)=number of identicalpositions/total number of positions×100). Preferably, the number andsize of the gaps, which are required to optimize the alignment of thetwo sequences, are taken into consideration.

The comparison and determination of the identity between two sequencescan be carried out by using a mathematical algorithm. A specific exampleof the mathematical algorithm that can be used for comparing thesequences includes an algorithm described in Karlin and Altschul (1990)Proc. Natl. Acad. Sci. USA 87:2264-68 and modified by Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. However, thealgorithm is not necessarily limited to this. Such an algorithm isincorporated in NBLAST program and XBLAST program (version 2.0)described in Altschul et al. (1990) J. Mol. Biol. 215: 403-10. In orderto obtain a nucleotide sequence equivalent to the nucleic acid moleculeof the present invention, for example, BLAST nucleotide search withscore=100 and word length=12 may be carried out by the NBLAST program.In order to obtain an amino acid sequence equivalent to the polypeptidemolecule of the present invention, for example, BLAST polypeptide searchwith score=50 and word length=3 may be carried out by the XBLASTprogram. In order to obtain gapped alignments for comparison, GappedBLAST described in Altschul et al., (1997) Amino Acids Research 25(17):3389-3402 can be utilized. In using BLAST and Gapped BLAST, the defaultparameters of the corresponding programs (e.g., XBLAST and NBLAST) canbe used. In detail, see http://www.ncbi.nlm.nih.gov. Another example ofthe mathematical algorithm that can be used for comparing sequencesincludes an algorithm described in Meyers and Miller (1988) Comput.Appl. Biosci. 4: 11-17. Such programs are incorporated into the ALIGNprogram that can be used for, for example, GENESTREAM network server(IGH Montpellier, France) or ISREC server. When the ALIGN program isused for comparison of the amino acid sequences, for example, PAM120weight residue table can be used in which a gap length penalty is 12 anda gap penalty is 4.

The identity between two amino acid sequences can be determined by usingthe GAP program in the GCG software package, using Blossom 62 matrix orPAM250 matrix with the gap weight of 12, 10, 8, 6, or 4, and the gaplength weight of 2, 3, or 4. Furthermore, the homology between twonucleic acid sequences can be determined using the GAP program in theGCG software package (available at http://www.gcg.com) with the gapweight of 50 and the gap length weight of 3.

The present enzyme may be a part of a larger protein (for example,fusion protein). Examples of a sequence to be added in the fusionprotein may include a sequence useful for purification, for example, asequence of a multi histidine residue, and an additional sequence forsecuring the safety for producing a recombinant, and the like.

The present enzyme having the above-mentioned amino acid sequence can beprepared easily by a genetic engineering technique. For example, thepresent enzyme can be prepared by transforming an appropriate host cell(for example, Escherichia coli) by DNA encoding the present enzyme, andby collecting proteins expressed in the transformant. The collectedproteins are appropriately purified according to the purposes. In thecase where the present enzyme is prepared as a recombinant protein,various modifications can be carried out. For example, DNA encoding thepresent enzyme and other appropriate DNA are inserted into the samevector and the vector is used for producing a recombinant protein. Then,the enzyme consisting of a recombinant protein to which arbitrarypeptide or protein is linked can be obtained. Furthermore, modificationmay be carried out so as to cause addition of sugar chain and/or lipidor processing of N-terminal or C-terminal. The above-mentionedmodification permits extraction of a recombinant protein, simplificationof purification, addition of biological functions, or the like.

(DNA Encoding β-Amylase)

A second aspect of the present invention provides a gene encoding thepresent enzyme, that is, a novel β-amylase gene. In one embodiment, thegene of the present invention includes DNA encoding the amino acidsequence set forth in SEQ ID NO: 7. A specific example of thisembodiment is a DNA consisting of the base sequence set forth in SEQ IDNO: 6.

In general, when a part of DNA encoding a certain protein is modified, aprotein encoded by the modified DNA may sometimes have the equalfunction to that of a protein encoded by the DNA before modification.That is to say, the modification of the DNA sequence does not have asubstantial effect on the function of the encoded protein, so that thefunction of the encoded protein may be maintained before and after themodification. Thus, as another embodiment, the present inventionprovides DNA encoding a protein having a base sequence equivalent to thebase sequence set forth in SEQ ID NO: 6 and having the β-amylaseactivity (hereinafter, which is also referred to as “equivalent DNA”).The “equivalent base sequence” herein denotes a base sequence which ispartly different from the base sequence set forth in SEQ ID NO: 6 but inwhich the function (herein, β-amylase activity) of the protein encodedby the sequence is not substantially affected by the difference.

A specific example of the equivalent DNA includes DNA that hybridizes tothe complementary base sequence of the base sequence of SEQ ID NO: 6under stringent conditions. Herein, the “stringent conditions” arereferred to as conditions in which a so-called specific hybrid is formedbut a nonspecific hybrid is not formed. Such stringent conditions areknown to persons skilled in the art. Such stringent conditions can beset with reference to, for example, Molecular Cloning (Third Edition,Cold Spring Harbor Laboratory Press, New York) and Current protocols inmolecular biology (edited by Frederick M. Ausubel et al., 1987). Anexample of the stringent conditions can include a condition in which ahybridization solution (50% formamide, 10×SSC (0.15 M NaCl, 15 mM sodiumcitrate, pH 7.0), 5× Denhardt solution, 1% SDS, 10% dextran sulfate, 10μg/ml denatured salmon sperm DNA, and 50 mM phosphate buffer (pH 7.5))is used and incubated at about 42° C. to about 50° C., thereafter,washed with 0.1×SSC and 0.1% SDS at about 65° C. to about 70° C. Furtherpreferable stringent conditions can include, for example, a condition inwhich a hybridization solution 50% formamide, 5×SSC (0.15 M NaCl, 15 mMsodium citrate, pH 7.0), 1× Denhardt solution, 1% SDS, 10% dextransulfate, 10 μg/ml denatured salmon sperm DNA, and 50 mM phosphate buffer(pH 7.5)) is used.

Another specific example of the equivalent DNA can include DNA encodinga protein having a base sequence which includes substitution, deletion,insertion, addition or inversion in one or a plurality of bases when thebase sequence of SEQ ID NO: 6 is a reference base sequence, and whichhas a β-amylase activity. The substitution, deletion, or the like, ofthe base may occur in a plurality of sites. The “plurality” hereindenotes, for example, 2 to 40 bases, preferably 2 to 20 bases, and morepreferably 2 to 10 bases, although it depends upon the positions ortypes of the amino acid residue in the three-dimensional structure ofthe protein encoded by the DNA. The above-mentioned equivalent DNA canbe obtained by modifying DNA having the base sequence shown in SEQ IDNO: 6 so as to include substitution, deletion, insertion, additionand/or inversion of base by using treatment with a restriction enzyme;treatment with exonuclease, DNA ligase, etc; introduction of mutation bya site-directed mutagenesis (Molecular Cloning, Third Edition, Chapter13, Cold Spring Harbor Laboratory Press, New York) and randommutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold SpringHarbor Laboratory Press, New York), and the like. Furthermore, theequivalent DNA can be also obtained by other methods such as irradiationwith ultraviolet ray.

A further example of the equivalent DNA can include DNA havingdifference in base as mentioned above due to polymorphism represented bySNP (single nucleotide polymorphism).

The gene of the present invention can be prepared in an isolated stateby using a standard genetic engineering technique, a molecularbiological technique, a biochemical technique, and the like, withreference to sequence information disclosed in the present specificationor attached sequence list. Specifically, the gene of the presentinvention can be prepared by appropriately using oligonucleotideprobe/primer capable of specifically hybridizing to the gene of thepresent invention from an appropriate genome DNA library or a cDNAlibrary of Bacillus flexus, or cell body extract of Bacillus flexus. Anoligonucleotide probe/primer can be easily synthesized by using, forexample, a commercially available automated DNA synthesizer. As to aproduction method of libraries used for preparing the gene of thepresent invention, see, for example, Molecular Cloning, Third Edition,Cold Spring Harbor Laboratory Press, New York.

For example, a gene having the base sequence set forth in SEQ ID NO: 6can be isolated by using a hybridization method using all or a part ofthe base sequence or its complimentary sequence as a probe. Furthermore,amplification and isolation can be carried out by using a nucleic acidamplification reaction (for example, PCR) using a synthesizedoligonucleotide primer designed to specifically hybridize to a part ofthe base sequence. Furthermore, it is possible to obtain a target geneby chemical synthesis based on the information of the amino acidsequence set forth in SEQ ID NO: 7 or the base sequence set forth in SEQID NO: 6 (see, reference document: Gene, 60(1), 115-127 (1987)).

Hereinafter, a specific example of the method of obtaining the gene ofthe present invention is described. Firstly, the present enzyme(β-amylase) is isolated and purified from Bacillus flexus, andinformation about the partial amino acid sequence is obtained. As amethod for determining the partial amino acid sequence thereof, forexample, purified β-amylase is directly subjected to amino acid sequenceanalysis [protein-sequencer 476A, Applied Biosystems] by EdmanDegradation [Journal of biological chemistry, vol. 256, pages 7990 -7997(1981)] according to a routine method. It is effective that limitedhydrolysis is carried out by allowing protein hydrolase to act, theobtained peptide fragment is separated and purified, and the thusobtained purified peptide fragment is subjected to the amino acidsequence analysis.

Based on the information of thus obtained partial amino acid sequence, aβ-amylase gene is cloned. Cloning can be carried out by using, forexample, a hybridization method or a PCR method. When the hybridizationmethod is used, for example, a method described in Molecular Cloning(Third Edition, Cold Spring Harbor Laboratory Press, New York) can beused.

When the PCR method is used, the following method can be used. Firstly,PCR reaction is carried out by using a synthesized oligonucleotideprimer designed based on the information of the partial amino acidsequence using a genome DNA of a microorganism producing β-amylase as atemplate, and thus a target gene fragment is obtained. The PCR method iscarried out according to the method described in PCR Technology, editedby Erlich. H A, Stocktonpress, 1989]. Furthermore, when a base sequenceis determined by a method usually used in the amplification DNAfragment, for example, a dideoxy chain terminator method, a sequencecorresponding to the partial amino acid sequence of β-amylase other thanthe sequence of the synthesized oligonucleotide primer is found in thedetermined sequence, and a part of the β-amylase gene can be obtained.When a hybridization method and the like is further carried out by usingthe obtained gene fragment as a probe, a gene encoding the full lengthof the β-amylase can be cloned.

In the below mentioned Examples, a sequence of a gene encoding β-amylaseproduced by Bacillus flexus is determined by using the PCR method. Thecomplete base sequence of a gene encoding β-amylase produced by Bacillusflexus is shown in SEQ ID NO: 6. Furthermore, the amino acid sequenceencoded by the base sequence is determined (SEQ ID NO: 7). In additionto the base sequence shown in SEQ ID NO: 6, a plurality of the basesequences corresponding to the amino acid sequence set forth in SEQ IDNO: 7 are present.

All or a part of the β-amylase gene (SEQ ID NO: 6) whose complete basesequence has been clarified is used as a probe of hybridization, andthereby DNA having high homology with respect to the β-amylase gene ofSEQ ID NO: 6 can be selected from a genome DNA library or a cDNA libraryof microorganisms producing other β-amylase.

Similarly, a primer for PCR can be designed. By carrying out PCRreaction using this primer, a gene fragment having high homology withrespect to the above-mentioned β-amylase gene can be detected and,furthermore, a complete gene thereof can be obtained.

Protein of the obtained gene is manufactured, and its β-amylase activityis measured. Thereby, it is possible to confirm whether or not theobtained gene is a gene encoding a protein having the β-amylaseactivity. Furthermore, by comparing the base sequence (or the amino acidsequence encoded thereby) of the obtained gene with the base sequence(or the amino acid sequence encoded thereby) of the above-mentionedβ-amylase gene, the gene structure or the homology may be examined,thereby determining whether or not the gene encodes protein having theβ-amylase activity.

Since the primary structure and the gene structure are clarified,modified β-amylase (a gene subjected to at least one of deletion,addition, insertion, and substitution of one or a plurality of aminoacid residues) can be obtained by introduction of random mutation orsite-specific mutation. This makes it possible to obtain a gene encodingβ-amylase that has a β-amylase activity but has different optimumtemperature, thermostability, optimum pH, stable pH, substratespecificity, and the like. Furthermore, it becomes possible tomanufacture modified β-amylase by genetic engineering.

Herein, a scheme for introducing mutation is carried out withconsideration of, for example, a characteristic sequence of a genesequence. The consideration of a characteristic sequence can be made byconsidering, for example, the prediction of the three-dimensionalstructure of the protein, and homology to existing proteins.

Examples of the method for introducing random mutation include: amethod, as method of chemically treating DNA, which causes transitionmutation in which sodium hydrogensulfite is allowed to act and cytosinebase is converted into uracil base [Proc. Natl. Acad. Sci. U.S.A., 79,1408-1412 (1982)]; a method, as a biochemical method, which causes basesubstitution during the process of synthesizing the double strand in thepresence of [α-S]dNTP [Gene, vol 64, pages 313-319 (1988)]; a method, asa method of using PCR, which carries out PCR in a reaction system withmanganese added, thereby lowering fidelity of incorporation ofnucleotides [Anal. Biochem., 224, 347-353 (1995)], and the like.

Examples of the method for introducing site-specific mutation include amethod using amber mutation [gapped duplex method; Nucleic Acids Res.,Vol. 12, No. 24, 9441-9456 (1984)]; a method using a recognition site ofthe restriction enzyme [Analytical Biochemistry, Vol. 200, pages 81-88(1992), Gene, Vol. 102, pages 67-70 (1991)]; a method using mutation ofdut (dUTPase) and ung (uracil-DNA glycosilase) [Kunkel method; Proc.Natl. Acad. Sci. U.S.A., 82, 488-492 (1985)]; a method using ambermutation using DNA polymerase and DNA ligase [Oligonucleotide-directedDual Amber: ODA) method, Gene, Vol. 152, pages 271-275 (1995), JapanesePatent Application Unexamined Publication No. H7-289262]; a method usinga host inducing a repair system of DNA (Japanese Patent ApplicationUnexamined Publication No. H8-70874); a method using a proteincatalyzing a DNA strand exchange reaction (Japanese Patent ApplicationUnexamined Publication No. H8-140685); a method by PCR using two typesof primers for introducing a restriction enzyme into which therecognition site is added (U.S. Pat. No. 5,512,463); a method by PCPusing a double strand DNA vector having inactivated drug-resistant geneand two types of primers [Gene, Vol. 103, pages 73-77 (1991)]; a methodby PCR using amber mutation [International Publication WO98/02535], andthe like.

The site-specific mutation can be easily introduced by usingcommercially available kits. Examples of the commercially available kitsinclude Mutan-G (register trade mark, Takara Shuzo Co., Ltd.) using thegapped duplex method, Mutan-K (register trade mark, Takara Shuzo Co.,Ltd.) using the Kunkel method, Mutan-ExpressKm (register trade mark,Takara Shuzo Co., Ltd.) using the ODA method, QuikChange™ Site-DirectedMutagenesis Kit [STRATAGENE] using a primer for introducing mutation andDNA polymerase derived from Pyrococcus furiosus, and the like.Furthermore, as the kits using the PCR method, for example, TaKaRa LAPCR in vitro Mutagenesis Kit (Takara Shuzo Co., Ltd.), Mutan (registertrade mark)—Super Express Km (Takara Shuzo Co., Ltd.), and the like.

Thus, the primary structure and the gene structure of β-amylase areprovided by the present invention. As a result, it is possible togenetically manufacture proteins having a β-amylase activity with highpurity at low cost.

(Recombinant Vector)

Another aspect of the present invention relates to a recombinant vectorcontaining the gene of the present invention. The term “vector” as usedin this specification is intended to refer to a nucleic acid moleculecapable of transporting nucleic acid that is inserted in the vector tothe inside of the target such as cells. The types or forms of vector arenot particularly limited. Therefor, examples of the vector may be in aform of a plasmid vector, a cosmid vector, a phage vector, a viralvector (e.g., an adenovirus vector, an adeno-associated virus vector, aretrovirus vector, a herpes virus vector, etc).

According to the purpose of use (cloning, protein expression), and byconsidering the types of host cells, an appropriate vector is selected.Specific examples of the vector include a vector using Escherichia colias a host (M13 phage or the modified body thereof, λ phage or themodified body thereof, pBR322 or the modified body thereof (pB325,pAT153, pUC8, etc.) and the like), a vector using yeast as a host(pYepSec1, pMFa, pYES2, etc.), a vector using insect cells as a host(pAc, pVL, etc.), a vector using mammalian cells as a host (pCDM8,pMT2PC, etc.), and the like.

The recombinant vector of the present invention is preferably anexpression vector. The term “expression vector” is a vector capable ofintroducing the nucleic acid inserted therein into the target cells(host cells) and being expressed in the cells. The expression vectorusually includes a promoter sequence necessary for expression of theinserted nucleic acid and an enhancer sequence for promoting theexpression, and the like. An expression vector including a selectionmarker can be used. When such an expression vector is used, by using theselection marker, the presence or absence of the introduction of anexpression vector (and the degree thereof) can be confirmed.

Insertion of the gene of the present invention into a vector, insertionof the selection marker gene (if necessary), and insertion of a promoter(if necessary), and the like, can be carried out by a standardrecombination DNA technology (see, for example, Molecular Cloning, ThirdEdition, 1.84, Cold Spring Harbor Laboratory Press, New York, aalready-known method using restriction enzyme and DNA ligase).

(Transformant)

The present invention further relates to a transformant into which thegene of the present invention is introduced. In the transformant of thepreset invention, the gene of the present invention exists as anexogenous molecule. Preferably, the transformant of the presentinvention can be preferably prepared by transfection or transformationusing the vector of the present invention mentioned above. Thetransfection and transformation can be carried out by, for example, acalcium phosphate coprecipitation method, electroporation (Potter, H. etal., Proc. Natl. Acad. Sci. U.S.A. 81, 7161-7165(1984)), lipofection(Felgner, P. L. et al., Proc. Natl. Acad. Sci. U.S.A. 84,7413-7417(1984)), microinjection (Graessmann, M. & Graessmann, A., Proc. Natl.Acad. Sci. U.S.A. 73,366-370 (1976)), a method by Hanahan (Hanahan, D.,J. Mol. Biol. 166, 557-580 (1983)), a lithium acetate method (Schiestl,R. H. et al., Curr. Genet. 16, 339-346 (1989)), protoplast—polyethyleneglycol method (Yelton, M. M. et al., Proc. Natl. Acad. Sci. 81,1470-1474 (1984)), and the like.

Examples of the host cell may include microorganism, animal cells, plantcells, and the like. Examples of microorganisms may include bacterialcells such as Escherichia coli, Bacillus sp., Streptomyces sp., andLactococcus sp.; yeast such as Saccharomyces sp., Pichia sp., andKluyveromyces sp.; filamentous fungi such as Aspergillus sp.,Penicillium sp., and Trichoderma sp. As the animal cell, baculovirus maybe used.

(Manufacturing Method of β-Amylase)

A further aspect of the present invention provides a manufacturingmethod of β-amylase. In one embodiment of the manufacturing method ofthe present invention, a step of culturing Bacillus flexus havingability of producing the present enzyme (β-amylase) (step (1)), and astep of collecting the β-amylase from a culture solution and/or a cellbody after culture (step (2)) are carried out.

Examples of Bacillus flexus to be used in the step (1) may include theabove-mentioned Bacillus flexus DSM1316, DSM1320, DSM1667, APC9451, andthe like. The culturing method and the culture conditions are notparticularly limited as long as the target enzyme is produced. That isto say, on the condition that the present enzyme is produced, a methodsand culture conditions suitable for culturing of microorganisms to beused can be set appropriately. As the culture method, any of liquidculture and solid culture may be employed, but liquid culture ispreferred. The culture conditions are described taking liquid culture asan example.

Any media can be used as long as microorganisms to be used can grow. Forexample, a medium containing a carbon source such as glucose, sucrose,gentiobiose, soluble starch, glycerin, dextrin, syrup, and organicacids; a nitrogen source such as ammonium sulfate, ammonium carbonate,ammonium phosphate, ammonium acetate, or peptone, yeast extract, cornsteep liquor, casein hydrolysate, bran, meat extract, and the like; andfurther, inorganic salts such as potassium salt, magnesium salt, sodiumsalt, phosphate salt, manganese salt, iron salt, and zinc salt, can beused. In order to promote the growth of microorganisms to be used,vitamin, amino acid, and the like may be added to the medium. The pH ofthe medium is adjusted to, for example, about 3 to 10, and preferably,about 7 to 8. The culturing temperature is generally about 10° C. to 50°C., and preferably about 20° C. to 37° C. The culturing is carried outfor one to seven days, preferably three to four days under aerobicconditions. As a culturing method, for example, a shake culture method,and an aerobic submerged culture method with a jar fermenter can beemployed.

After the culturing in the above-mentioned conditions, β-amylase iscollected from the culture solution or the cell body (step (2)). Whenβ-amylase is collected from the culture solution, the present enzyme canbe obtained by separation and purification after removing insolublematters by, for example, filtration, centrifugation of culturesupernatant followed by carrying out any combinations of concentrationby ultrafiltration, salting out of ammonium sulfate precipitation,dialysis, various types of chromatography such as ion-exchange resin,and the like.

On the other hand, when the present enzyme is collected from the cellbody, the present enzyme can be obtained by pulverizing the cell body bypressuring treatment, ultrasonication, and the like, followed byseparation and purification thereof similar to the above. Note here thatthe above-mentioned series of processes (pulverizing, separation, andpurification of cell body) may be carried out after the cell body iscollected from a culture solution by filtration, centrifugation, and thelike.

Note here that confirmation of expression or confirmation of expressionproduct can be carried out easily by using an antibody againstβ-amylase, but expression can be confirmed by measuring the β-amylaseactivity.

According to another embodiment of the present invention, β-amylase ismanufactured by using the above-mentioned transformant. In themanufacturing method in this embodiment, firstly, the above-mentionedtransformant is cultured in the conditions in which the protein encodedby the introduced gene is produced (step (i)). As to various vector-hostsystems, the culture conditions for transformant are well-known, and aperson skilled in the art can set appropriate culture conditions easily.After the culturing step, a step of collecting the produced protein(i.e., β-amylase) is carried out (step (ii)). Collection and thefollowing purification may be carried out by the same method asmentioned in the above-mentioned embodiment. The purification degree ofthe present enzyme is not particularly limited. Furthermore, the finalform may be a liquid state or a solid state (including a powder state).

(Enzyme Preparation)

The enzyme of the present invention is provided in a form of, forexample, an enzyme preparation. The enzyme preparation may contain, inaddition to an active ingredient (the enzyme of the present invention),excipient, buffer agents, suspension agents, stabilizer, preservatives,antiseptics, physiologic saline, and the like. Examples of the excipientmay include lactose, sorbitol, D-mannitol, sucrose, and the like.Examples of the buffer agent may include phosphate, citrate, acetate,and the like. Examples of the stabilizer may include propylene glycol,and ascorbic acid, and the like. Examples of the preservative mayinclude phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol,methyl paraben, and the like. Examples of the antiseptic may includebenzalkonium chloride, parahydroxybenzoate, chlorobutanol, and the like.

(Application of β-Amylase)

A further aspect of the present invention provides a method of producingmaltose as an application of β-amylase derived from Bacillus flexus. Inthe production method according to the present invention, β-amylasederived from Bacillus flexus is allowed to act on a substrate consistingof polysaccharide or oligosaccharide having α-1,4 linkage of glucose asa main chain. Examples of the substrate may include soluble starch,potato starch, corn starch, amylopectin, glycogen, andmaltooligosaccharide. The purity of the substrate is not particularlylimited. Therefore, β-amylase may be allowed to act on the substrate ina state in which it is mixed with other materials. Furthermore,β-amylase may be allowed to act on two or more substratessimultaneously.

The production method of the present invention is characterized by usingβ-amylase derived from Bacillus flexus. Preferably, as the β-amylase,the above-mentioned β-amylase (the present enzyme) of the presentinvention is used.

The production method of the present invention is used for producing,for example, maltose-containing syrup, maltose starch syrup, and thelike. The production method of the present invention may be used forimproving the quality of bread or antioxidant means for rice-cake andrice-cake sweets.

Example <Measurement Method of β-Amylase Activity>

The activity of β-amylase was measured as follows. To 0.5 ml of 0.1Mphosphate-HCl buffer solution (pH 5.0) containing 1% soluble starch and10 mM calcium acetate, 0.5 ml of enzyme solution was added. The mixturewas incubated at 37° C. for 30 minutes. Then, 2.5 ml of DNS solution(0.2% DNS, 80 mM NaOH, 0.2 M potassium sodium tartrate tetrahydrate) wasadded to the mixture so as to stop the reaction. After the reaction wasstopped, the reaction mixture was boiled for five minutes, theabsorbance was measured at the wavelength of 530 nm. The enzyme amountwhen the absorbance measured at the wavelength of 530 nm is 1 is definedas one unit.

1. Confirmation of β-Amylase Derived from Bacillus flexus

Four strains of Bacillus flexus, DSM1316, DSM1320, DSM1667, and APC9451were cultured with shaking at 30° C. for three days by using a liquidmedium containing the compositions shown in Table 1.

TABLE 1 β-amylase production medium (w/v) Corn steep liquor 2% Solublestarch 4% Calcium carbonate 2%

The β-amylase activity in the resultant culture supernatant was measuredby the above-mentioned measurement method of the β-amylase activity. Theresults are shown in Table 2.

TABLE 2 Activity DSM1316 4.0 DSM1320 14.8 DSM1667 4.0 APC9451 5.72. Production and Purification of β-Amylase Derived from Bacillus flexusAPC9451

Bacillus flexus APC9451 was cultured with shaking at 30° C. for threedays by using a liquid medium having the compositions shown in Table 1.The obtained culture supernatant solution was four-times concentrated byusing an UF membrane (AIP-0013, Asahi Kasei Corporation), and thenammonium sulfate was added thereto so as to obtain 60% saturationconcentration. The precipitate fraction was dissolved again in 20 mMacetic acid buffer solution (pH 5.5). Then, to the mixture solution,ammonium sulfate was added so as to obtain 20% saturation concentration.The resultant precipitates were removed by centrifugation, and thensubjected to HiPrep Butyl 16/10 FF column (GE Healthcare) that had beenequilibrated with 20 mM acetic acid buffer solution (pH 5.5) containingammonium sulfate with 20% saturation concentration. Then, adsorbedβ-amylase protein was eluted by linear concentration gradient ofammonium sulfate from 20% saturation concentration to 0% saturationconcentration.

The collected β-amylase activity fractions were concentrated by using anUF membrane, and then subjected to HiTrap CM FF column (GE Healthcare)that had been equilibrated with 20 mM acetic acid buffer solution (pH5.5). The adsorbed β-amylase protein was eluted by linear concentrationgradient of 0 M to 0.5 M of NaCl.

Furthermore, the collected β-amylase activity fractions wereconcentrated by using an UF membrane, and then subjected to HiLoad 16/60Superdex200 column (GE Healthcare) that had been equilibrated with 20 mMacetic acid buffer solution (pH 5.5) containing 0.15 M NaCl, and elutedwith the same buffer solution. The β-amylase activity fractions werecollected and subjected to desalting and concentration by usingultrafiltration membrane, and thus purified enzyme preparation wasobtained. The resultant purified enzyme was examined for the belowmentioned properties. Furthermore, the enzyme was subjected toN-terminal amino acid sequence analysis and the internal peptide aminoacid sequence analysis.

Note here that results of purification in each stage are shown in Table3. The specific activity in the final stage was 2270 times as comparedwith that of the crude enzyme. FIG. 5 shows the results of SDS-PAGE (CBBstaining) with 10-20% gradient gel of samples of each step in thepurification process. It is shown that the purified enzyme preparation(lane 4) is a single protein in SDS-PAGE.

TABLE 3 Total protein Total activity Specific Collection amount (mg) (U)activity (u/mg) rate (%) Concentrated 27200 18700 0.69 100 solutionAmmonium 2856 9054 3.17 48 sulfate fractionation Butyl FF 59.9 4120 68.822 CM FF 0.64 656 1031 4 Superdex200 0.084 132 1569 1

3. Various Properties of Thermostable β-Amylase (1) Optimum ReactionTemperature

According to the above-mentioned β-amylase activity measurement method,reaction was carried out at the reaction temperatures of 25° C., 37° C.,50° C., 55° C., 60° C., 65° C., and 70° C. The values are shown as therelative activity when a value at a temperature exhibiting the maximumactivity is defined as 100%. The optimum reaction temperature was around55° C. (FIG. 1).

(2) Optimum Reaction pH

For the substrate, 1% soluble starch was used, and the measurement wascarried out in each buffer solution (citric acid buffer: pH2, pH3, andpH4, Britton-Robinson buffer: pH4, pH5, pH6, pH7, pH8, pH9, pH10, andpH11) at 37° C. for 10 minutes. The values are shown as the relativeactivity when pH exhibiting the maximum activity value is defined as100%. The optimum reaction pH was about 8.0 (FIG. 2).

(3) Thermostability

The enzyme solution (20 u/ml) was subjected to heat treatment in 0.1 Macetic acid—hydrochloric acid buffer solution (pH 5.0) containing 10 mMcalcium acetate at each temperature of 37° C., 50° C., 55° C., 60° C.,65° C. and 70° C. for 10 minutes, and then the remaining activity wasmeasured by the above-mentioned β-amylase activity measurement method.The values are shown as the remaining activity when the remainingactivity with no heat treatment is defined as 100%. The heat treatmentat 55° C. for 10 minutes shows the remaining activity of 90% or more.The thermostability was shows until 55° C. (FIG. 3).

(4) pH Stability

β-amylase was treated in each of the buffer solutions (citric acidbuffer: pH2, pH3, and pH4, Britton-Robinson buffer: pH4, pH5, pH6, pH7,pH8, pH9, pH10, and pH11) at 30° C. for three house, and then theactivity was measured by the above-mentioned β-amylase activitymeasurement method. The values are shown as the relative activity whenpH exhibiting the maximum activity value is defined as 100%. The optimumreaction pH was 4 to 9 (FIG. 4).

(5) Molecular Weight Measurement by SDS-PAGE

SDS-PAGE was carried out according to the method by Laemmli et al. Notehere that molecular weight marker used was Low Molecular WeightCalibration Kit for Electrophoresis (GE Healthcare), which includedPhosphorylase b (97,000 Da), Albumin (66,000 Da), Ovalbumin (45,000 Da),Carbonic anhydrase (30,000 Da), Trypsin inhibitor (20,100 Da), anda-Lactalbumin (14,400 Da) as a reference protein. Electrophoresis wascarried out at 20 mA/gel for 80 minutes by using a gradient gel (Wako)having a gel concentration of 10-20%, and the molecular weight wasobtained. As a result, the molecular weight was about 60 kDa (FIG. 5).

(6) Isoelectric Point

When the isoelectric point of the present enzyme was measured byisoelectric point precipitation using Ampholine (electrification at600V, at 4° C., for 48 hours), it was 9.7.

(7) Effect of Metal Ion and Inhibitor

To β-amylase in 0.1 M acetic acid—hydrochloric acid buffer solution (pH5.0) containing 10 mM calcium acetate, 1 mM of various metal ions orinhibitor were added, respectively, treated at 30° C. for 30 minutes,and then the activity was measured by the above-mentioned β-amylaseactivity. The results are shown in Table 4. The values were shown as therelative activity when the metal ion and inhibitor were not added isdefined as 100%. The activity was inhibited by Cu ion, iodine aceticacid, PCMB, and SDS.

TABLE 4 Relative activity Na⁺ 88 K⁺ 96 Ca²⁺ 130 Mn²⁺ 222 Mg²⁺ 103 Zn²⁺96 Cu²⁺ 46 Fe²⁺ 105 Fe³⁺ 113 EDTA 97 N-ethylmaleimide 93 PCMB 25monoiodoacetic acid 14 SDS 37 No additives 100

(8) Substrate Specificity

The β-amylase activity with respect to each substrate was examined. Thevalues are shown as the relative activity when the activity with respectto soluble starch is defined as 100%. As to oligosaccharides, theproduction amount of maltose was examined by the below-mentionedquantification amount of maltose. After 0.1 u/ml enzyme was reacted to0.5% of each maltooligosaccharide at 37° C. for 30 minutes,quantification of maltose was carried out by HPLC (Aminex carbohydrateHPX-42A, BIO-RAD). A production amount of maltose when the substrate issoluble starch is defined as 100%, the relative activity with respect toeach maltooligosaccharide was calculated from the production amount ofmaltose.

The results are shown in Table 5. The values were shown as the relativeactivity when a production amount of maltose with respect to solublestarch is defined as 100%. Cyclodextrin, pullulan, and dextran werealmost broken down. Oligosaccharide did not act on maltotriose but wellacted on the other oligosaccharides.

TABLE 5 Substrate Relative activity (%) Maltotriose 0 Maltotetraose 75Maltopentaose 102 Maltohexaose 131 Maltoheptaose 111 α-cyclodextrin 0β-cyclodextrin 1.4 γ-cyclodextrin- 0.6 Amylose 98 Amylopectin 83Pullulan 3.4 Dextran, 1.9 Glycogen 51 Potato starch 78 Corn starch 85Waxy corn starch 106 Soluble starch 1004. Obtaining Gene Fragment Encoding β-Amylase Derived from Bacillusflexus

(a) Isolation of Chromosome DNA

Genome DNA was prepared from a cell body of Bacillus flexus obtained in“1” by the Saito-Miura method (Biochim. Biophys. Acta, 72, 619-629,1963).

(b) Determination of Partial Amino Acid Sequence

The purified preparation of β-amylase obtained in “1” was subjected toamino acid sequence analysis so as to determine the N-terminal aminoacid sequence (SEQ ID NO: 1) and internal peptide amino acid sequence(SEQ ID NOs: 2 and 3) of 10 residues.

(c) Preparation of DNA Probe by PCR

Two types of mixed oligonucleotides were synthesized based on theN-terminal amino acid sequence and the internal amino acid sequence toobtain a PCR primer (SEQ ID NOs: 4 and 5). By using these primers andchromosome DNA of Bacillus flexus as a template, PCR reaction wascarried out in the following conditions.

<PCR Reaction Solution>

10× PCR reaction buffer solution (TaKaRa): 5.0 μl

dNTP mixture solution (2.5 mM each, TaKaRa): 4.0 μl

25 mM MgCl₂: 5 μl

100 μM sense primer: 3.0 μl

100 μM antisense primer: 3.0 μl

Distilled water: 28.5 μl

Chromosome DNA solution (140 μg/ml): 1 μl

LA Taq DNA polymerase (TaKaRa): 0.5 μl

<PCR Reaction Conditions>

Stage 1: denaturation (94° C., 5 minutes) 1 cycle

Stage 2: denaturation (94° C., 30 seconds) 30 cycles

Annealing (48° C., 30 seconds)

Elongation (72° C., 1 minute)

About 0.86 kb of the obtained DNA fragment was cloned to pGEM-Teasy(Promega), and then the base sequence was confirmed. In immediatelyafter the sense primer and immediately before the antisense primer, thebase sequence encoding the above-mentioned partial amino acid sequencewas found. This DNA fragment was defined as a DNA probe for full lengthgene cloning.

(d) Production of Gene Library

As a result of Southern hybridization analysis of chromosome DNA ofBacillus flexus, about 5.0 kb of single band that hybridizes to a probeDNA in KpnI decomposition product was confirmed. In order to clone thisKpnI DNA fragment of about 5.0 kb, a gene library was produced asfollows. Chromosome DNA prepared by the above-mentioned (a) wassubjected to KpnI treatment. 28 μg of genome DNA of Bacillus flexus, 3μl of 10× L buffer solution, 26 μl of distilled water, and 1 μl of KpnIwere mixed and treated at 37° C. for 15 hours. The obtaineddecomposition product was ligated to pUC19 (TaKaRa) vector that had beensubjected to KpnI treatment so as to obtain a gene library.

(e) Screening of Gene Library

The DNA fragment (0.86 kb) that had been obtained in the above-mentioned(c) was labeled with DIG-High Prime (Roche). This was used as a DNAprobe, and then the gene library that had been obtained in (d) wassubjected to screening by colony hybridization. A plasmid pUC19-BAF wasobtained from the resultant positive colony.

(f) Determination of Base Sequence

The base sequence of the plasmid pUC19-BAF was determined according to aroutine method. The base sequence (1638 bp) encoding β-amylase is shownin SEQ ID NO: 6. Furthermore, the amino acid sequence (545 amino acids)encoded by SEQ ID NO: 6 is shown in SEQ ID NO: 7. In this amino acidsequence, the amino acid sequence in the N-terminal region (SEQ IDNO: 1) and the internal amino acid sequences (SEQ ID NOs: 2 and 3),which had been determined in (b), were found.

5. Expression of β-Amylase Derived from Bacillus flexus in Escherichiacoli

(a) Structure of Expression Plasmid of β-Amylase in Escherichia coli

Two types of oligonucleotides (SEQ ID NOs: 8 and 9) were synthesizedbased on the DNA sequence encoding the amino acid sequences in theN-terminal region and the C-terminal region, PCR primers were obtained.To the sense primer, the NdeI restriction enzyme recognition site wasadded, and to the antisense primer, the XhoI restriction enzymerecognition site was added. The PCR reaction was carried out in thefollowing conditions by using these primers and a plasmid pUC19-BAFhaving a β-amylase gene as a template.

<PCR Reaction Solution>

-   10× PCR reaction buffer solution (TOYOBO) 5.0 μl-   dNTP mixture solution (2.5 mM each, TOYOBO) 5.0 μl-   10 μM sense primer: 1.5 μl-   10 μM antisense primer: 1.5 μl-   25 mM MgSO₄ 2 μl-   Distilled water: 33 μl-   Plasmid pUC19-BAF solution (83 μg/ml): 1.0 μl-   KOD-Plus-DNA polymerase (TOYOBO): 1.0 μl

<PCR Reaction Conditions>

Stage 1: denaturation (94° C., 2 minutes) 1 cycle

Stage 2: denaturation (94° C., 15 seconds) 30 cycles

Annealing (60° C., 30 seconds)

Elongation (68° C., 2 minutes)

The obtained PCR product was confirmed by electrophoresis. Then, it waspurified with GENE CLEANE III (34 μl), and 4 μl of 10×H buffer solutionand 1 μl of NdeI and 1 μl of XhoI were added. The mixture was subjectedto enzyme treatment at 37° C. for 15 hours. The restriction enzymetreatment solution was confirmed by electrophoresis and purified.Thereafter, the mixture was ligated to a vector pET20(b) (TAKARA BIOINC) that had been treated with NdeI and XhoI in advance, and thus theexpression plasmid pET-BAF was obtained (FIG. 6). Furthermore, whetheror not the base sequence encoding β-amylase in pET-BAF was correct wasconfirmed.

(b) Expression of β-Amylase in Escherichia coli

The expression plasmid pET-BAF was introduced into Escherichia coli BL21(DE3) (Novagen). From the transformants obtained as an ampicillinresistant strain, a strain holding pET-BAF in which the target β-amylasegene had been introduced by colony PCR was selected. Furthermore, atransformant of Escherichia coli BL21 (DE3) having an expression vectorpET20(b) was obtained as a control. These transformants were cultured in4 ml of LB medium containing 50 μg/ml ampicillin at 18° C., at 160 rpmfor 47 hours, and cells were collected. The cell bodies were suspendedin 0.5 ml of 20 mM acetic acid buffer solution (pH 5.5), to which 0.25 gof glass beads with φ0.1 mm were added, the cell bodies were disruptedby using a multi-beads shocker (Yasui Kikai). As the disruptioncondition, 3.5 cycles of 30 seconds ON and 30 seconds OFF were repeated.The obtained cell free-extract was centrifuged to obtain a solublecomponents.

The activities of the obtained samples were measured according to theabove-mentioned β-amylase activity measurement method. The results areshown in Table 6.

TABLE 6 Activity (U/ml) Protein (mg/ml) Specific activity (U/mg) pET-BAF43.5 7.9 5.5 pET20(b) 0.4 8.0 0.05

INDUSTRIAL APPLICABILITY

The β-amylase of the present invention has a thermostability that iscomparable to the β-amylase derived from soybeans, and is suitable forapplications that require the reaction at high temperatures. The use ofthe β-amylase of the present invention makes it possible to carry outthe enzyme reaction at high temperatures that are less susceptible tocontamination of various bacteria. Therefore, the β-amylase of thepresent invention is particularly useful in application such as foodprocessing and saccharification.

The present invention is not limited to the description of the aboveembodiments and Examples. A variety of modifications, which are withinthe scopes of the following claims and which are easily achieved by aperson skilled in the art, are included in the present invention.

Contents of the theses, Publication of Patent Applications, PatentPublications, and other published documents referred to in thisspecification are herein incorporated by reference in its entity.

1. A β-amylase derived from Bacillus flexus.
 2. A β-amylase having thefollowing enzymological properties: (1) action: acting on α-1,4glucoside linkage of polysaccharides and oligosaccharides to liberatemaltose; (2) substrate specificity: acting well on starch, amylose,amylopectin, glycogen, maltotetraose, maltopentaose, maltohexaose, andmaltoheptaose, but not acting on pullulan, dextran, cyclodextrin, andmaltotriose; (3) optimum temperature: about 55° C.; (4) optimum pH:about 8.0; (5) thermostability: stable at 55° C. or lower (pH 5.0, 10minutes); (6) pH stability: stable at pH 4 to 9 (30° C., three hours);and (7) molecular weight: about 60,000 (SDS-PAGE).
 3. A β-amylase havingan amino acid sequence set forth in SEQ ID NO: 7, or an amino acidsequence equivalent to the amino acid sequence.
 4. The β-amylaseaccording to claim 3, wherein the equivalent amino acid sequence is anamino acid sequence having about 90% or more identity to the amino acidsequence set forth in SEQ ID NO:
 7. 5. An enzyme preparation comprisingβ-amylase according to claim 1 as an active ingredient.
 6. A β-amylasegene comprising DNA selected from the group consisting of the following(A) to (C): (A) DNA encoding an amino acid sequence set forth in SEQ IDNO: 7; (B) DNA having a base sequence set forth in SEQ ID NO: 6; and (C)DNA having a base sequence equivalent to the base sequence set forth inSEQ ID NO: 6, and having a β-amylase activity.
 7. A recombinant vectorcontaining a β-amylase gene according to claim
 6. 8. A transformant intowhich a β-amylase gene according to claim 6 is introduced.
 9. Amanufacturing method of β-amylase, the method comprising the followingsteps (1) and (2) or steps (i) and (ii): (1) culturing Bacillus flexushaving an ability of producing β-amylase: (2) collecting β-amylase froma culture solution and/or a cell body after culturing; (i) culturing atransformant described in claim 8 under conditions in which a proteinencoded by the gene is produced; and (ii) collecting the producedprotein.
 10. The manufacturing method according to claim 9, whereinBacillus flexus is a strain specified by the accession number NITEBP-548.
 11. A Bacillus flexus strain specified by the accession numberNITE BP-548.
 12. A production method of maltose, the method comprisingallowing β-amylase derived from Bacillus flexus to act on polysaccharideor oligosaccharide having α-1,4 linkage of glucose as a main chain. 13.The production method comprising allowing β-amylase derived fromBacillus flexus to act on polysaccharide or oligosaccharide having α-1,4linkage of glucose as a main chain, wherein the β-amylase is a β-amylaseaccording to claim
 2. 14. An enzyme preparation comprising β-amylaseaccording to claim 2 as an active ingredient.